Cardiovascular diseases are one of the leading causes of death worldwide. It is estimated that every year, about 12 million people throughout the world die due to cardiovascular diseases. Cardiovascular diseases are considered to be very serious diseases that cost the United States US$ 260 billion every year, but no effective treatment has yet been identified.
Recently, studies have been reported on inducing the differentiation of human embryonic stem cells into vascular endothelial cells, muscle cells, etc., raising hope for the possibility of treatment of cardiovascular diseases using human embryonic stem cells.
Human embryonic stem cells retain totipotency that is the ability to differentiate into three germ cell layers (endodermal, ectodermal, mesodermal) which organize the human body. Human embryonic stem cells can be differentiated into specific cells according to their surrounding environment, and thus, are expected to become potent tools that can achieve significant progress in the medical and science fields. Thus, it is expected that studies of human embryonic stem cells can provide important clues for primitive aspects of early stages of human differentiation and can play a critical role in studies of cell therapy for cardiovascular diseases and incurable diseases, such as Parkinson's disease, myocardial infarction, diabetes, and leukemia.
Human embryonic stem cells can be obtained by isolating and culturing the inner cell mass of an early-stage human embryo known as “blastocyst”. Human embryonic stem cells retain totipotency, and at the same time, can be maintained in an undifferentiated state and can be continuously sub-cultured (Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A, Swiergiel J J, Marshall V S, Jones J M, Embryonic stem cell lines derived from human blastocysts. Science (1998) 282:1145-1147). Thus, if conditions for differentiation into specific cells, proliferation, isolation, and recovery are developed and established, cell therapy studies are expected to face a major turning point. In this regard, many studies about human embryonic stem cells have focused on establishing differentiation conditions that can induce the differentiation of human embryonic stem cells into specific cells, such as neural cells, vascular endothelial cells, cardiac cells, endothelial cells, and hepatocytes.
Differentiated embryoid bodies (EBs) include large amounts of other differentiated cell lineages and some undifferentiated cells, in addition to target cells. Thus, a technique of effectively isolating only target cells after differentiation is essentially required. However, satisfactory methods capable of selectively and efficiently isolating only target cells have not yet been reported.
Zhang S C et al. reported a method for isolating neural progenitor cells from differentiated embryoid bodies by dispase treatment (Zhang S C, Thomson J A et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature Biotech (2001) 19, 1129-1133). However, Zhang S C et al's report is related to isolation of neural progenitor cells and is silent about whether or not the isolation method can be applied to other types of differentiated cells, e.g., vascular endothelial cells.
In connection with differentiation and isolation of vascular endothelial cells from human embryonic stem cells, a method of differentiating human embryonic stem cells into embryoid bodies and isolating vascular endothelial cells from the embryoid bodies using a Fluorescence Activated Cell Sorter (FACS) has been reported (Levenberg S, Golub J S, Amit M, Itskovitz-Eldor J, Langer R. PNAS (2002) 99, 4391-4396). However, while isolating single cells from differentiated embryoid bodies using FACS, large amounts of the cells are destroyed, and further, the recovery rate of vascular endothelial cells is merely 2%.